Antimicrobial peptide, process to obtain a peptide and uses therefor

ABSTRACT

The invention refers to small peptides with low hemolytic activity, presenting equal antiparasitic, antifungal and antibacterial activities. More specifically, it refers to a peptide called gomesin, with 18 amino acid residues, configured as a clamp-like structure consisting of two anti-parallel beta-folded sheets joined by a beta turn, containing four invariable residues of cystein forming two disulphide bridges, optionally configurable as a cyclic chain with closed edges.

[0001] The invention refers to small peptides with low hemolyticactivity, presenting balanced activity against parasites, fungi andbacteria.

[0002] A number of peptides extracted from animals and plants have shownactivity against infection. The application PCT WO9503325, published onFeb. 2, 1995, mentions peptides called protegrins, also reviewingliterature on this subject, which includes references on tachyplesins,poliphemusins, defensins, β-defensins and insect defensins. The U.S.Pat. No. 5,994,306 refers to more specific protegrins.

[0003] The application PCT WO9702287, published on Jan. 23, 1997,discloses peptides called parevins and tachytegrins, which are similarto protegrins, except for cysteins on positions 6 and 15.

[0004] This invention has the object to disclose new and small peptideswhich are similar in some aspects to protegrins, tachytegrins andparevins, but present balanced anti-parasitic, anti-bacteria andanti-fungus activity, besides low hemolitic activity, as well asprocesses to obtain them and their applications. This is a peptidecalled gomesin, configured as a clamp-like structure consisting of twoanti-parallel beta-folded sheets, joined by a beta turn, containing fourinvariable cysteine residues forming two disulphide bridges, with thefollowing general formula (1):

[0005] in which:

[0006] Z₁ is:

[0007] in the absence of X₁₉, a free amino end residue or a residue witha blocked end amino group by methylation, carbamylation, acylation,acetylation, and some protecting groups like terbutyl, etc., beingpreferrably pyroglutamic acid, providing resistance against proteaseactivity;

[0008] when X₁₉ is present, a basic, hydrophobic, polar/large or smallresidue, with a free amino end group available to close the edges of themolecule, such as glutamine or glutamic acid.

[0009] C₂, C₆, C₁₁ and C₁₅ are independently cystein, homocystein orpennicilamine;

[0010] P₁ and P₂ are disulphide bridges from C₂ to C₁₅, and from C₆ toC₁₁, respectively, either one or both bridges being present;

[0011] A₃, A₄, A₅, A₁₂, A₁₃, A₁₄, A₁₆, A₁₇ are independently basic,hydrophobic, polar/large or small residues, provided that:

[0012] A₃, A₄ and A₁₆ are preferably basic amino acid residues,

[0013] A₅, A₁₂ and A₁₄ are preferably hydrophobic residues; and

[0014] A₁₇ is preferably a small residue,

[0015] A₇ to A₁₀ are residues which are able to effect a beta turn andcan be independently basic, hydrophobic, polar/large or small, with atleast one of them being basic and the other hydrophobic, with A₈ and A₁obeing preferably basic amino acid residues,

[0016] A₁₈ is:

[0017] in the absence of X₁₉: a basic, hydrophobic, polar/large or smallresidue, preferably a basic residue, bearing a free carboxyl group orforming an acceptable salt such as potassium, sodium, calcium, magnesiumor other with an organic or inorganic ion, or amidated with an amine ofthe formula NH₃ or RNH₂ or R₂NH, in which R is independently a saturatedor insaturated hydrocarbyl with one to six carbons, such as methyl,ethyl, isopropyl, t-butyl, n-pentyl, cyclohexyl, 2-cyclohexenyl,3-cyclohexenyl, 4-hexinyl and similar;

[0018] when X₁₉ is present: a basic, hydrophobic, polar/large or smallresidue, preferably a basic residue, which free carboxyl group end isinvolved with the closing of peptide molecule edges

[0019] X₁₉ may be absent or present; if present, it is a chemical linkbetween Z₁ and A₁₈ or a chemical structure or molecule present betweenZ₁ and A₁₈ is and linked to both, closing the amino acid chain edges ofthe peptide of the invention;

[0020] from about 15% to about 50% of all amino acid residues should bebasic;

[0021] they should present a positive net charge of at least +1 atphysiological pH.

[0022] Some of the peptides of the invention can be obtained byextraction from animals, such as from the spider Acanthoscurriagomesiana, and due to this origin they are called gomesins. They canalso be synthetically produced and, when containing only geneticallycoded amino acids, they can be produced in a recombinant way. Peptidesof the invention are useful for the treatment and prevention of animaland plant infection as caused by parasites, bacteria and/or fungi. Inanother aspect, DNA coding peptides of the invention may be expressed insitu, in animals or plants, to fight infection. Peptides of theinvention can also be used as standard for antimicrobial tests and forbinding to endotoxins.

[0023] Peptides of the invention can be obtained in a recombinant way bymeans of peptide-coding cDNA expression in heterologous systems, as wellknown in the literature.

[0024] The invention also refers to useful compositions againstbacteria, fungi and parasites, used in the combat against suchorganisms.

[0025] Peptides of the invention are generally different from othersknown in the art, among other reasons, for presenting the followingqualities which had not been simultaneously found so far:

[0026] small structure, being therefore prone to be better distributedwithin tissues and less immunogenic;

[0027] they are about equally active against bacteria, fungi andparasites (while e.g. protegrins are less efficient against fungi);

[0028] they have low hemolytic activity, especially in a closed edgecyclic configuration.

[0029] Peptides of the invention contain a beta turn connecting twobeta-turned sheets. As it is known by one skilled in the art, a betafold refers tipically to a peptide segment containing residues of fouramino acids reverting the amino acid chain direction. Cysteins onpositions 2, 6, 11 and 15 notably provide for the existence of the betaturn by forming dissulphide bridges between them, i.e. from C₂ to C₁₅,and from C₆ to C₁₁.

[0030] As known in the literature, dissulphide bridges can besubstituted by lactam bridges or any other bridge playing an equivalentrole.

[0031] In an alternative embodiment, peptides of the invention canpresent a cyclic structure with closed edges in the peptide chain.Methods to obtain closed cyclic peptides is known in the art.

[0032] In non-cyclic peptides, as known to one skilled in the art, aminoand carboxy ends may be derived. In the peptides of the invention, theamino group end may be methyled, carbamyled, acyled, acetylated or aspyroglutamic acid. Peptides of the invention may, by means of additionto the carboxyl end of the molecules, be present as inorganic salts,such as chloride, bromide, iodide, fluoride, sulphate, nitrate,phosphate, etc., or organic salts, such as acetate, formate, benzoate,etc. The acceptance of each one of the above salts depends on thedesired use, which is routinely understood. The carboxyl end can also beamidated. Derivation reactions are known.

[0033] As mentioned in this description, amino acid residues of thepeptides of the invention are defined under the followingcharacteristics:

[0034] acid: the residue has a negative charge due to the loss of the Hion under physiologic pH, and the residue is attracted to an acquoussolution, so to search for superficial positions in the configuration ofa peptide in which it is contained, provided it is in acquous media withphysiologic pH;

[0035] basic: the residue has a positive charge due to its associationwith the H ion under physiologic pH, and the residue is attracted to anacquous solution, so to seek superficial positions in the configurationof a peptide in which it is contained, provided that it is in acquousmedia with physiologic pH;

[0036] hydrophobic: the residue is not charged under physiologic pH andis repelled by an acquous solution, so to seek more internal positionsin the configuration of a peptide in which it is contained, providedthat the peptide is in acquous media;

[0037] polar/large: the residue is not charged under physiologic pH, butit is not sufficiently repelled by an acquous solution, so that it couldseek more internal positions in the configuration of a peptide in whichit is contained, provided that the peptide is in acquous media;

[0038] polar/small: neutral residue, since its side chains are notsufficiently large, even if polar groups are absent, to make them becomehydrophobic. It contains four or less carbons when at least one polargroup appears in the side chain and three or less carbons when this isnot the case.

[0039] Concerning amino acids of naturally-occurring proteins, as usedin the terms of this application, the following classification is used:

[0040] acids: aspartic acid, glutamic acid

[0041] basic:

[0042] non cyclic: arginine, lysine

[0043] cyclic: histidine

[0044] small: glycine, serine, alanine, threonine

[0045] polar/large: asparagine, glutamine

[0046] hydrophobic: tyrosine, valine, isoleucine, leucine, methyonine,phenyl alanine, tryptophan.

[0047] The functional equivalents of the peptide of the invention arealso the said compounds where one or several aminoacids are enantiomers,diastereoisomers, natural aminoacids with a D-conformation, unusualaminoacids such as Ca—methyl—aminoacid, hydroxylysine, methyllysine,dimethyllysine and preferentially pyroglutamic acid at the particularposition of the residue A1 at the N-terminus of the said compounds. Theinvention is also covering the retropeptides and theretro-inversopeptides and the synthetic aminoacids such as theornithine, the norleucine, the cyclohexyl-alanine and omega-aminoacids.

[0048] Cystein and other amino acid residues containing sulphydryl-SHare not contemplated in the above list, since their capacitiy to formdissulphide bridges providing for a secondary structure is critical forthe compounds of the invention.

[0049] Without creating limits to the scope of the invention, it isunderstood that the action of the peptide of the invention againstmicroorganisms and parasites is enhanced by a larger number ofhydrophobic amino acid residues.

[0050] According to some embodiments, constituents of the generalformula (1) of the peptide of the invention are as follows:

[0051] a) in the absence of X₁₉:

[0052] Z₁ is pyroglutamic acid;

[0053] A₃, A₄, A₈, A₁₀ and A₁₆ are basic residues, with preferably A₃,A₄, A₁₀ and A₁₆ being arginine and A₈ being lysine;

[0054] A₁₈ is a basic residue, preferably amidated arginine, optionallynon-amidated;

[0055] A₅, A₇, A₁₂ and A₁₄ are hydrophobic residues, with preferably A₅being leucine, A₇ and A₁₄ being tyrosine and A₁₂ being valine;

[0056] A₉ is a polar/large residue, preferably glutamine;

[0057] A₁₃ and A₁₇ are small residues, preferably with A₁₃ beingthreonine and A₁₇ being glycine.

[0058] b) in the presence of X₁₉, all components of the peptide of theinvention are the same as above, except that:

[0059] Z₁ is a basic amino acid, hydrophobic, polar/large or smallresidue with a free amino group end, e.g. glutamine.

[0060] A₁₈ is a basic residue, with a free carboxyl end group involvedin closing peptide edges.

[0061] X₁₉ is a chemical structure present between Z₁ and A₁₈, connectedto both so to close the peptide chain edges.

EXAMPLES OF PROCESSES TO OBTAIN THE PEPTIDES OF THE INVENTION

[0062] The examples given herein are intended to explain the invention,and do not add any limitation to the claims that follow at the end ofthis specification.

[0063] 1) Extraction of Gomesin From the Spider Acanthoscurriagomesiana.

[0064] By means of the process as described below, a gomesincorresponding specifically to the following structure within the generalformula (1) is obtained, including two disulphide bridges (schematicspace representation just for illustrative purposes):

[0065] in which:

[0066] Z=pyroglutamic acid

[0067] C=cystein

[0068] R=arginine

[0069] L=leucine

[0070] Y=tyrosine

[0071] K=lysine

[0072] Q=glutamine

[0073] V=valine

[0074] T=threonine

[0075] G=glycine

[0076] R_(a)=arginine with an amidated carboxyl group end

[0077] Hemolymph (approximately 0.4 ml/spider) of both male and femaleanimals in different development stages was collected from pre-cooledanimals by heart puncture with an apyrogenic syringe, in the presence ofsodium citrate buffer (30 mmol/L, pH 4.6) containing NaCl (450 mmol/L),EDTA (10 mmol/L) and glucose (100 mmol/L). Hemocytes are removed fromplasma by centrifugation at 800×g for 10 minutes at 4° C. Entirehemocytes are washed once with sodium citrate buffer and lysated byconcentration in a vacuum centrifuging machine.

[0078] After concentration, hemocytes are re-suspended in a 1.5 ml of 2Macetic acid supplemented with aprotinin (20 μg/ml) as proteaseinhibitor, being homogenized in a Dounce equipment (maximum 152μ,minimum 76μ). A second homogenization is effected by means of sonication(3×30s) at average intensity, kept in an ice cold water bath and theextraction is effected for 30 minutes at 4° C. under mild stiring. Thesupernatant obtained by means of centrifugation at 13,800×g for 30minutes at 6° C. is directly submitted to pre-purification by solidphase extraction.

[0079] Organella and cytosolic acid extracts are applied to solid phasecolumns connected in series, balanced in acidic water (0.05%trifluoroacetic acid). Three elutions are successively effected with 5%,40% and 80% acetonitrile in acidic water. The 40% acetonitrile portionis concentrated by centrifugation under vacuum, reconsistuted with MiliQwater and reverse phase chromatographed in a column which is balancedwith 2% acetonitrile in acidified water. Elution was performed with alinear gradient of acetonitrile from 2 to 60% in acidified water for 120minutes, at a flow rate of 1.3 ml/min.

[0080] The active fraction against the tested bacterium Micrococcusluteus of hemocytes (AGH2) is additionally purified by filtrationchromatography. Elution is made under isocratic conditions with 30%acetonitrile in acidic water at a flow rate of 0.4 ml/min.

[0081] HPLC (high pressure liquid chromatography) purification is madeat room temperature. The effluent column is monitored for its absorbanceat 225 nm. Fractions corresponding to absorbance peaks are collected,concentrated under vacuum and reconstituted in MilliQ water.Anti-bacterial activity of the collected fractions was monitored by aliquid growth inhibition assay according to J. Biol. Chem., 268,14893-14897 (1993), using Micrococcus luteus as the test microorganism.

[0082] 2) Gomesin Synthesis

[0083] Peptides of the invention have been synthesized using a classicFmoc procedure as described in J. Biol. Chem., 271, 29537-29544 (1996).

[0084] For comparison with the gomesin obtained by extraction, asmentioned before, the following peptide was synthesized:

[0085] where asterisks inidcates a C-terminal α-amide.

[0086]FIG. 1 attached shows a table regarding the activity spectrum forgomesin, both amidated (Example 1)and non-amidated (Example 2), againstbacteria, fungi and yeast. The evaluation of activity against bacteriaand fungi was done as described in J. Biol. Chem., 271, 29537-29544(1996).

[0087] On this table, n. d. means “non-detected” for the testedconcentration range of up to 100 μM for gomesin and 50 μM forandroctonin, while n. m. means not measured.

[0088] The table shows that the peptides of the invention haveanti-bacteria properties. As shown, gomesin (from Example 1 above) iseffective against most tested strains, with 24 from the 27 strains beingsusceptible to gomesin under MIC (minimum inhibitory concentration)below 1.6 μM for half of them, under concentrations from 1.6 to 6.25 μM(42%). Anti-bacterial properties have also been verified in the peptidesof the invention, e.g. by the mortality of Micrococcus luteus andEntamoeba coli D22 as shown in FIG. 2. 10 μM of the gomesin of Example 1(solid line) or water (dotted line) have been added to an exponentialstage culture of M. luteus (circles) or E. coli (triangles). Aliquoteswere removed at different time intervals and the number of CFU (colonyforming units) counted by plating on Luria Bertani agar plates afterovernight incubation at 37° C. The kinetic of killing of gomesin, asshown after approximately one minute, is one of the advantages of theinvention.

[0089] Anti-Parasitic Activity

[0090] As an example of anti-parasitic activity, the cell viability ofLeishmania (leishmania) amazonensis (MRPO/BR/72/M 1841-LV-79) wasevaluated, by meaking use of the MTT test[3-(4,5-dimethyltiazolyl-2)-2,5-diphenyl tetrazolio bromide] asdescribed in J Immunol. Methods 65,55-63 (1983) and adapted forLeishmania spp, according to J. Immunol. Methods 127,11-18 (1990).

[0091] Quantities from 0.1 to 100 μM have been tested against theparasite Leishmania (leishmania) amazonensis. After incubation for onehour, the viability of the parasite was noticed as dependent on the usedconcentration of gomesin, as shown in FIG. 3. Synthetic gomesin ofExample 1 shown as solid bars, and a fragment of hemoglobin (33-61bovine alpha-hemoglobin) shown as hatched bars and used as a positivecontrol, has been incubated with the parasites for one hour at 22° C.under concentrations from 0.3 to 80 μM. The viability of the parasite(%) has been measured by making use of the MTT assay as described in J.Immunol. Methods 65, 55-63 (1983) and adapted for Leishmania spp,according to J. Immunol. Methods 127, 11-18 (1990).

[0092] Antibodies

[0093] Antibodies to the peptides of the invention can be produced bymaking use of standard immunologic technics for the production ofpoliclonal antibodies and, if required, by perpetuating antibodyproducing cells of the immunized host as a source for the production ofmonoclonal antibodies.

[0094] Gomesin-Containing Formulations

[0095] Peptides of the invention may be used in various compositionswhich are active against bacteria, fungi and parasites. They can be usedindividually, as a mixture with other peptides of the invention, withother microbicidal agents or both, and jointly with other ingredientsknown in the art, e.g. active principles such as erythromicine,tetracycline, azithromicine, cephalosporines, etc. and generalconstituents such as carriers, diluents, excipients, etc.

[0096] Peptides of the invention may be formulated for pharmaceutical orveterinary use.

[0097] The formulations of the invention can be presented in any form,adapted to the intended purpose as well known by an expert in the art,e.g. for topic use such as creams, oils, ointments, powders, gels, etc.or appropriately for oral, transdermal, transmucous, intramuscular,intravenous, subcutaneous, etc. administration.

1 100 1 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 1 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr TyrCys Arg Gly Arg 1 5 10 15 2 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 2 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 3 18 PRT Arachinid BINDING(1)..(18) 3 Xaa Cys Lys Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr CysArg Gly Arg 1 5 10 15 4 18 PRT Arachinid BINDING (1)..(18) 4 Xaa Cys HisArg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 518 PRT Arachinid BINDING (1)..(18) 5 Xaa Cys Arg Lys Leu Cys Tyr Lys GlnArg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 6 18 PRT Arachinid BINDING(1)..(18) 6 Xaa Cys Arg His Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr CysArg Gly Arg 1 5 10 15 7 18 PRT Arachinid BINDING (1)..(18) 7 Xaa Cys ArgArg Tyr Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 818 PRT Arachinid BINDING (1)..(18) 8 Xaa Cys Arg Arg Val Cys Tyr Lys GlnArg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 9 18 PRT Arachinid BINDING(1)..(18) 9 Xaa Cys Arg Arg Ile Cys Tyr Lys Gln Arg Cys Val Thr Tyr CysArg Gly Arg 1 5 10 15 10 18 PRT Arachinid BINDING (1)..(18) 10 Xaa CysArg Arg Met Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 1015 11 18 PRT Arachinid BINDING (1)..(16) 11 Xaa Cys Arg Arg Phe Cys TyrLys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 12 18 PRTArachinid BINDING (1)..(18) 12 Xaa Cys Arg Arg Trp Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 13 18 PRT Arachinid BINDING(1)..(18) 13 Xaa Cys Arg Arg Leu Cys Leu Lys Gln Arg Cys Val Thr Tyr CysArg Gly Arg 1 5 10 15 14 18 PRT Arachinid BINDING (1)..(18) 14 Xaa CysArg Arg Leu Cys Val Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 1015 15 18 PRT Arachinid BINDING (1)..(18) 15 Xaa Cys Arg Arg Leu Cys IleLys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 16 18 PRTArachinid BINDING (1)..(18) 16 Xaa Cys Arg Arg Leu Cys Met Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 17 18 PRT Arachinid BINDING(1)..(18) 17 Xaa Cys Arg Arg Leu Cys Phe Lys Gln Arg Cys Val Thr Tyr CysArg Gly Arg 1 5 10 15 18 18 PRT Arachinid BINDING (1)..(18) 18 Xaa CysArg Arg Leu Cys Trp Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 1015 19 18 PRT Arachinid BINDING (1)..(18) 19 Xaa Cys Arg Arg Leu Cys TyrArg Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 20 18 PRTArachinid BINDING (1)..(18) 20 Xaa Cys Arg Arg Leu Cys Tyr His Gln ArgCys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 21 18 PRT Arachinid BINDING(1)..(18) 21 Xaa Cys Arg Arg Leu Cys Tyr Lys Asn Arg Cys Val Thr Tyr CysArg Gly Arg 1 5 10 15 22 18 PRT Arachinid BINDING (1)..(18) 22 Xaa CysArg Arg Leu Cys Tyr Lys Gln Lys Cys Val Thr Tyr Cys Arg Gly Arg 1 5 1015 23 18 PRT Arachinid BINDING (1)..(18) 23 Xaa Cys Arg Arg Leu Cys TyrLys Gln His Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 24 18 PRTArachinid BINDING (1)..(18) 24 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Leu Thr Tyr Cys Arg Gly Arg 1 5 10 15 25 18 PRT Arachinid BINDING(1)..(18) 25 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Tyr Thr Tyr CysArg Gly Arg 1 5 10 15 26 18 PRT Arachinid BINDING (1)..(18) 26 Xaa CysArg Arg Leu Cys Tyr Lys Gln Arg Cys Ile Thr Tyr Cys Arg Gly Arg 1 5 1015 27 18 PRT Arachinid BINDING (1)..(18) 27 Xaa Cys Arg Arg Leu Cys TyrLys Gln Arg Cys Met Thr Tyr Cys Arg Gly Arg 1 5 10 15 28 18 PRTArachinid BINDING (1)..(18) 28 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Phe Thr Tyr Cys Arg Gly Arg 1 5 10 15 29 18 PRT Arachinid BINDING(1)..(18) 29 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Trp Thr Tyr CysArg Gly Arg 1 5 10 15 30 18 PRT Arachinid BINDING (1)..(18) 30 Xaa CysArg Arg Leu Cys Tyr Lys Gln Arg Cys Val Gly Tyr Cys Arg Gly Arg 1 5 1015 31 18 PRT Arachinid BINDING (1)..(18) 31 Xaa Cys Arg Arg Leu Cys TyrLys Gln Arg Cys Val Ser Tyr Cys Arg Gly Arg 1 5 10 15 32 18 PRTArachinid BINDING (1)..(18) 32 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Ala Tyr Cys Arg Gly Arg 1 5 10 15 33 18 PRT Arachinid BINDING(1)..(18) 33 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Leu CysArg Gly Arg 1 5 10 15 34 18 PRT Arachinid BINDING (1)..(18) 34 Xaa CysArg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Val Cys Arg Gly Arg 1 5 1015 35 18 PRT Arachinid BINDING (1)..(18) 35 Xaa Cys Arg Arg Leu Cys TyrLys Gln Arg Cys Val Thr Ile Cys Arg Gly Arg 1 5 10 15 36 18 PRTArachinid BINDING (1)..(18) 36 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Met Cys Arg Gly Arg 1 5 10 15 37 18 PRT Arachinid BINDING(1)..(18) 37 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Phe CysArg Gly Arg 1 5 10 15 38 18 PRT Arachinid BINDING (1)..(18) 38 Xaa CysArg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Trp Cys Arg Gly Arg 1 5 1015 39 18 PRT Arachinid BINDING (1)..(18) 39 Xaa Cys Arg Arg Leu Cys TyrLys Gln Arg Cys Val Thr Tyr Cys Lys Gly Arg 1 5 10 15 40 18 PRTArachinid BINDING (1)..(18) 40 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys His Gly Arg 1 5 10 15 41 18 PRT Arachinid BINDING(1)..(18) 41 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr CysArg Thr Arg 1 5 10 15 42 18 PRT Arachinid BINDING (1)..(18) 42 Xaa CysArg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ser Arg 1 5 1015 43 18 PRT Arachinid BINDING (1)..(18) 43 Xaa Cys Arg Arg Leu Cys TyrLys Gln Arg Cys Val Thr Tyr Cys Arg Ala Arg 1 5 10 15 44 18 PRTArachinid BINDING (1)..(18) 44 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Lys 1 5 10 15 45 18 PRT Arachinid BINDING(1)..(18) 45 Xaa Cys Lys His Ile Cys Phe Lys Asn Arg Cys Trp Ser Val CysLys Gly Arg 1 5 10 15 46 18 PRT Arachinid BINDING (1)..(18) 46 Xaa CysArg His Leu Cys Tyr Lys Asn Lys Cys Val Thr Tyr Cys Arg Gly Lys 1 5 1015 47 18 PRT Arachinid BINDING (1)..(18) 47 Xaa Cys Arg Arg Val Cys TyrArg Gln Arg Cys Val Gly Ile Cys His Thr Arg 1 5 10 15 48 18 PRTArachinid BINDING (1)..(18) 48 Xaa Cys His Lys Met Cys Leu Lys Gln HisCys Tyr Ala Trp Cys Arg Gly Arg 1 5 10 15 49 18 PRT Arachinid BINDING(1)..(18) 49 Xaa Cys His Arg Leu Cys Ile His Gln Arg Cys Val Thr Tyr CysLys Ala Lys 1 5 10 15 50 18 PRT Arachinid BINDING (1)..(18) 50 Xaa CysArg Arg Tyr Cys Met Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 1015 51 18 PRT Arachinid BINDING (1)..(18) 51 Xaa Cys His Lys Phe Cys LeuLys Asn Lys Cys Phe Ser Tyr Cys Lys Thr Arg 1 5 10 15 52 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 52 XaaCys Lys Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 510 15 53 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 53 Xaa Cys His Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr TyrCys Arg Gly Xaa 1 5 10 15 54 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 54 Xaa Cys Arg Lys Leu Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 55 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 55 Xaa Cys Arg His Leu CysTyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 56 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 56 XaaCys Arg Arg Tyr Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 510 15 57 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 57 Xaa Cys Arg Arg Val Cys Tyr Lys Gln Arg Cys Val Thr TyrCys Arg Gly Xaa 1 5 10 15 58 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 58 Xaa Cys Arg Arg Ile Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 59 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 59 Xaa Cys Arg Arg Met CysTyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 60 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 60 XaaCys Arg Arg Phe Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 510 15 61 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 61 Xaa Cys Arg Arg Trp Cys Tyr Lys Gln Arg Cys Val Thr TyrCys Arg Gly Xaa 1 5 10 15 62 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 62 Xaa Cys Arg Arg Leu Cys Leu Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 63 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 63 Xaa Cys Arg Arg Leu CysVal Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 64 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 64 XaaCys Arg Arg Leu Cys Ile Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 510 15 65 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 65 Xaa Cys Arg Arg Leu Cys Met Lys Gln Arg Cys Val Thr TyrCys Arg Gly Xaa 1 5 10 15 66 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 66 Xaa Cys Arg Arg Leu Cys Phe Lys Gln ArgCys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 67 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 67 Xaa Cys Arg Arg Leu CysTrp Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 68 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 68 XaaCys Arg Arg Leu Cys Tyr Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 510 15 69 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 69 Xaa Cys Arg Arg Leu Cys Tyr His Gln Arg Cys Val Thr TyrCys Arg Gly Xaa 1 5 10 15 70 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 70 Xaa Cys Arg Arg Leu Cys Tyr Lys Asn ArgCys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 71 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 71 Xaa Cys Arg Arg Leu CysTyr Lys Gln Lys Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 72 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 72 XaaCys Arg Arg Leu Cys Tyr Lys Gln His Cys Val Thr Tyr Cys Arg Gly Xaa 1 510 15 73 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 73 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Leu Thr TyrCys Arg Gly Xaa 1 5 10 15 74 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 74 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Tyr Thr Tyr Cys Arg Gly Xaa 1 5 10 15 75 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 75 Xaa Cys Arg Arg Leu CysTyr Lys Gln Arg Cys Ile Thr Tyr Cys Arg Gly Xaa 1 5 10 15 76 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 76 XaaCys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Met Thr Tyr Cys Arg Gly Xaa 1 510 15 77 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 77 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Phe Thr TyrCys Arg Gly Xaa 1 5 10 15 78 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 78 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Trp Thr Tyr Cys Arg Gly Xaa 1 5 10 15 79 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 79 Xaa Cys Arg Arg Leu CysTyr Lys Gln Arg Cys Val Gly Tyr Cys Arg Gly Xaa 1 5 10 15 80 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 80 XaaCys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Ser Tyr Cys Arg Gly Xaa 1 510 15 81 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 81 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Ala TyrCys Arg Gly Xaa 1 5 10 15 82 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 82 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Leu Cys Arg Gly Xaa 1 5 10 15 83 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 83 Xaa Cys Arg Arg Leu CysTyr Lys Gln Arg Cys Val Thr Val Cys Arg Gly Xaa 1 5 10 15 84 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 84 XaaCys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Ile Cys Arg Gly Xaa 1 510 15 85 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 85 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr MetCys Arg Gly Xaa 1 5 10 15 86 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 86 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Phe Cys Arg Gly Xaa 1 5 10 15 87 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 87 Xaa Cys Arg Arg Leu CysTyr Lys Gln Arg Cys Val Thr Trp Cys Arg Gly Xaa 1 5 10 15 88 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 88 XaaCys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Lys Gly Xaa 1 510 15 89 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 89 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr TyrCys His Gly Xaa 1 5 10 15 90 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 90 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln ArgCys Val Thr Tyr Cys Arg Thr Xaa 1 5 10 15 91 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 91 Xaa Cys Arg Arg Leu CysTyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ser Xaa 1 5 10 15 92 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 92 XaaCys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ala Xaa 1 510 15 93 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 93 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr TyrCys Arg Gly Xaa 1 5 10 15 94 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 94 Xaa Cys Lys His Ile Cys Phe Lys Asn ArgCys Trp Ser Val Cys Lys Gly Xaa 1 5 10 15 95 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 95 Xaa Cys Arg His Leu CysTyr Lys Asn Lys Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 96 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 96 XaaCys Arg Arg Val Cys Tyr Arg Gln Arg Cys Val Gly Ile Cys His Thr Xaa 1 510 15 97 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine orpenicillamine 97 Xaa Cys His Lys Met Cys Leu Lys Gln His Cys Tyr Ala TrpCys Arg Gly Xaa 1 5 10 15 98 18 PRT Arachinid DISULFID (2)..(15) C orhomocysteine or penicillamine 98 Xaa Cys His Arg Leu Cys Ile His Gln ArgCys Val Thr Tyr Cys Lys Ala Xaa 1 5 10 15 99 18 PRT Arachinid DISULFID(2)..(15) C or homocysteine or penicillamine 99 Xaa Cys Arg Arg Tyr CysMet Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 100 18 PRTArachinid DISULFID (2)..(15) C or homocysteine or penicillamine 100 XaaCys His Lys Phe Cys Leu Lys Asn Lys Cys Phe Ser Tyr Cys Lys Thr Xaa 1 510 15

1. Peptide of the general formula:

in which: Z₁ is: in the absence of X₁₉, a free amino end residue or aresidue with a blocked end amino group, providing resistance againstprotease activity; when X₁₉ is present, a basic, hydrophobic,polar/large or small residue, with a free amino end group available toclose the edges of the molecule. C_(2,) C_(6,) C₁₁ and C₁₅ areindependently cystein, homocystein or pennicilamine; P₁ and P₂ aredisulphide bridges from C₂ to C₁₅ and from C₆ to C₁₁, respectively,either one or both bridges being present; A₃, A₄, A₅, A₁₂, A₁₃, A₁₄,A₁₆, A₁₇ are independently basic, hydrophobic, polar/large or smallresidues; A₇ to A₁₀ are residues which are able to effect a beta turnand can be independently basic, hydrophobic, polar/large or small, withat least one of them being basic and the other hydrophobic; A₁₈ is: inthe absence of X₁₉: a basic, hydrophobic, polar/large or small residue,bearing a free carboxyl group end or forming an acceptable salt, oramidated with an amine of the formula NH₃ or RNH₂ or R₂NH, in which R isindependently a saturated or insaturated hydrocarbyl with one to sixcarbons; when X₁₉ is present: a basic, hydrophobic, polar/large or smallresidue, which free carboxyl end is involved with the closing of peptidemolecule edges; X₁₉ may be absent or present; if present, it is achemical link between Z₁ and A₁₈ or a chemical structure or moleculepresent between Z₁ and A₁₈ and linked to both, closing the amino acidchain edges of the peptide of the invention; from about 15% to about 50%of all amino acid residues are basic; the peptides present a positivecharge of at least +1 at physiologic pH.
 2. Peptide of claim 1 in which,in the absence of X₁₉, Z₁ is pyroglutamic acid.
 3. Peptide of claim 1 inwhich, when X₁₉ is present, Z₁ is a glutamine residue.
 4. Peptide ofclaim 1, in which A₃, A₄, A₈, A₁₀ and A₁₆ are basic amino acid residues.5. Peptide of claim 1, in which A₅, A₇, A₁₂ and A₁₄ are hydrophobicamino acid residues.
 6. Peptide of claim 1, in which A₁₃ and A₁₇ aresmall amino acid residues.
 7. Peptide of claim 1, in which A₈ and A₁₀are both basic amino acid residues.
 8. Peptide of claim 1 in which, inthe absence of X₁₉, A₁₈ is a basic amino acid residue.
 9. Peptide ofclaim 1 in which, when X₁₉ is present, A₁₈ is a basic amino acid residuewhich free carboxyl group end is involved in closing the edges of thepeptide molecule.
 10. Peptide of claim 4, in which the basic amino acidresidue is chosen from the group consisting of arginine, lysine andhistidine.
 11. Peptide of claim 5, in which the hydrophobic amino acidresidue is chosen from the group consisting of leucine, valine,isoleucine, methyonine, phenylalanine, tryptophan and tyrosine. 12.Peptide of claim 1, in which the A₉ residue is polar, preferably chosenfrom the group consisting of glutamine and asparagine.
 13. Peptide ofclaim 1, in which the small amino acid residues are chosen from thegroup consisting of threonine, glycine, serine and alanine.
 14. Peptideof claim 1, in which: A₃, A₄, A₁₀, A₁₆ and A₁₈ are arginine; A₅ isleucine; A₇ and A₁₄ are tyrosine; A₈ is lysine; A₉ is glutamine; A₁₂ isvaline; A₁₃ is threonine; A₁₇ is glycine; A₁₈ is amidated arginine; Z₁is pyroglutamic acid; X₉ is not present.
 15. Peptide of claim 1, whichis a cyclic chain with closed edges, in which: A₃, A₄, A₁₀, A₁₆ and A₁₈are arginine; A₅ is leucine; A₇ and A₁₄ are tyrosine; A₈ is lysine; A₉is glutamine; A₁₂ is valine; A₁₃ is threonine; A₁₇ is glycine; A₁₈ isarginine; Z₁ is glutamic acid; X₉ is a link or chemical structurelinking Z₁ to A₁₈.
 16. PROCESS TO OBTAIN A PEPTIDE by means ofextraction from arachnids, consisting of the stages of: collection ofhemolymph; separation of hemocytes from plasma; obtaining an acidextract from hemocytes; isolation of the peptide, preferably by HPLC;17. PROCESS TO OBTAIN PEPTIDE of claim 16, wherein said spider is anAcanthoscurria gomesiana.
 18. PROCESS TO OBTAIN A PEPTIDE of the generalformula

of claim 1, in which the Fmoc chemical synthesis techniques is used. 19.FORMULATION CONSISTING OF A PEPTIDE for the combat against infection inhuman beings, animals, and plants, especially for the combat againstbacteria, fungi and parasites, consisting, among other constituents, ofthe peptide of the general formula:

of claim
 1. 20. RECOMBINANT EXPRESSION SYSTEM for the production of apeptide of the general formula

of claim 1, in which said expression system consists of the nucleotidesequence coding the said peptide as linked to a controlling sequenceregulating said expression.
 21. METHOD TO PREVENT THE DEVELOPMENT OFPARASITES, FUNGI AND BACTERIA, in which said parasites, fungi andbacteria are contacted with an effective amount of a peptide of claim 1.22. METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIAof claim 21, in which the parasite is preferably a protozoary. 23.METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA ofclaim 21, in which the parasite is preferably a Leishmania.
 24. METHODTO INACTIVATE THE ENDOTOXIN FROM GRAM-NEGATIVE BACTERIA, in which saidendotoxin is contacted by an amount of a peptide of claim 1 which issubstantially effective to inactivate said endotoxin.
 25. ANTIBODY whichis substantially especifically reactive to the peptide of claim 1.